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Originally published In Press as doi:10.1074/jbc.M606516200 on August 28, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31605-31615, October 20, 2006
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Polysialic Acid Profiles of Mice Expressing Variant Allelic Combinations of the Polysialyltransferases ST8SiaII and ST8SiaIV*

Sebastian P. Galuska{ddagger}, Imke Oltmann-Norden§, Hildegard Geyer{ddagger}, Birgit Weinhold§, Klaus Kuchelmeister, Herbert Hildebrandt§, Rita Gerardy-Schahn§, Rudolf Geyer{ddagger}, and Martina Mühlenhoff§1

From the Institutes of {ddagger}Biochemistry and Neuropathology, Faculty of Medicine, University of Giessen, D-35392 Giessen, Germany and the §Institute of Cellular Chemistry, Hannover Medical School, D-30625 Hannover, Germany

Received for publication, July 10, 2006 , and in revised form, August 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo- and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of poly-Sia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate ~90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Carbohydrate modifications of proteins and lipids play an important role in the development and maintenance of the nervous system as mediators of cell recognition events (1). One striking example is polysialic acid (polySia),2 a linear homopolymer of {alpha}2,8-linked N-acetylneuraminic acid. In vertebrates, polySia is found almost exclusively as a post-translational modification of the neural cell adhesion molecule (NCAM), a member of the immunoglobulin superfamily. Attachment of polySia to NCAM was demonstrated to double the hydrodynamic radius of NCAM, thereby increasing the intermembrane space and disrupting the adhesive properties of NCAM and other cell adhesion molecules such as L1, integrins, and cadherins (2-4). PolySia promotes migration of neuronal precursor cells, axonal outgrowth, and synaptic plasticity (for review, see Ref. 5). In addition to its function as a negative regulator of cell adhesion, polySia was shown to bind heparan sulfate proteoglycans (6), forming a complex that supports synaptogenesis and activity-dependent remodeling of synapses (7). In addition, polySia can bind brain-derived neurotrophic factor to enhance brain-derived neurotrophic factor-dependent survival of cortical neurons (8, 9) and appears to be involved in the regulation of neurotransmitter receptor activity (10). Whereas polySia levels are high during embryonic development, expression in the adult is restricted to brain areas of persistent neurogenesis and synaptic plasticity (11).

Interestingly, the biosynthesis of polySia depends on two enzymes, the Golgi-resident polysialyltransferases ST8SiaII and ST8SiaIV, which share 59% identity at the amino acid sequence level. Each enzyme is independently capable of synthesizing polySia on NCAM, starting on complex N-glycans in the fifth Ig-like domain (12-15). During development, the enzymes are differentially expressed in a tissue- and cell type-specific manner with overlapping expression patterns (16-20). Studies to define distinct roles for each enzyme were performed in vitro using soluble forms of ST8SiaII and ST8SiaIV. In the in vitro situation, ST8SiaII was found to synthesize shorter polymers than ST8SiaIV (21, 22), and both enzymes together were described to act synergistically, yielding polySia chains with a higher degree of polysialylation (21, 23).

Genetic mouse models lacking either ST8SiaII (24) or ST8SiaIV (25) show only partial loss of polySia and mild but clearly distinct phenotypes. We recently generated polySia-deficient mice by simultaneous deletion of both polysialyltransferase genes (26). The dramatic phenotype observed in the double knock-out animals is characterized by postnatal growth retardation, hydrocephalus, defects in major brain fiber tracts, and precocious death (80% die within the first 4 weeks after birth). Most important, the lethal phenotype can be completely rescued by additional deletion of NCAM, demonstrating that polySia is a key regulator of NCAM functions (26).

To gain further insight into how each polysialyltransferase contributes to the biosynthesis of this essential carbohydrate structure, we intercrossed ST8SiaII and ST8SiaIV knock-out mice to generate offspring of all possible allelic combinations. Brain samples from a total of nine genotypes (including wildtype and double knock-out) were isolated at postnatal day 1 (P1) and compared with respect to the NCAM polysialylation status. In this study, we reveal the first in vivo picture of the complex NCAM polysialylation machinery as generated by the coordinated activities of ST8SiaII and ST8SiaIV.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, Enzymes, and Reagent—NCAM-specific mouse monoclonal antibody (mAb) KD11 (27) and polySia-specific mAb 735 (28) were used after affinity purification on protein G- and protein A-Sepharose (GE Healthcare), respectively. Endosialidase from bacteriophage K1F was purified as described previously (29). 1,2-Diamino-4,5-methylenedioxybenzene (DMB) was purchased from Dojindo, colominic acid from Sigma, and {alpha}2,8-linked sialic acid pentamers from Nacalai Tesque.

Breeding of Knock-out Mice—ST8SiaII (24) and ST8SiaIV (25) knock-out mice were backcrossed to C57BL/6J mice for six generations. The different genotypes used in this study were obtained by interbreeding heterozygous single knock-out strains or by crossing female ST8SiaII+/- ST8SiaIV-/- and male ST8SiaII-/- ST8SiaIV+/- mice. Genotyping was performed as described (26).

Protein Extraction and Western Blot Analysis—P1 mouse brains were homogenized (26), and one aliquot of each lysate was treated with 25 ng/µl endosialidase for 45 min on ice. Proteins were separated by 7% SDS-PAGE under reducing conditions, loading 40 µg of total protein/lane. Proteins were transferred to nitrocellulose and subjected to immunostaining using 2.5 µg/ml anti-polySia mAb 735 or anti-NCAM mAb KD11 and enhanced chemiluminescence for detection. The intensity of protein bands was analyzed on appropriately exposed Lumi-Film chemiluminescent detection films (Roche Applied Science) as the mean gray value by computerized densitometric scanning using Kodak 1D 3.5 Network software.

Immunohistochemistry—Freshly dissected mouse brains were fixed for 24 h with 4% paraformaldehyde in phosphate-buffered saline. Brains of wild-type, ST8SiaII-null, and ST8SiaIV-null mice were embedded in a single paraffin block and cut into 5-µm serial sections. Staining with mAb 735 (40 µg/ml in phosphate-buffered saline and 0.1% bovine serum albumin) was performed overnight at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Dako) and staining using an 3-amino-9-ethylcarbazole staining kit (Sigma). Sections were counterstained for 5 s with hematoxylin. For negative controls, serial brain sections were pretreated with endosialidase (3 µg/ml in phosphate-buffered saline and 0.1% bovine serum albumin) for 2 h at 37 °C before staining with mAb 735.

RNA Extraction and Quantitative Reverse Transcription (RT)-PCR—Total RNA (1 µg) isolated by TRIzol (Invitrogen) was transcribed with 200 units of SuperScript II reverse transcriptase (Invitrogen). PCR was performed in a final volume of 25 µl containing 240 nM each primer, 0.625 units of Platinum Taq DNA polymerase (Invitrogen), 200 µM dNTPs in Platinum Taq reaction buffer, 2 µl of SYBR Green I (1:10,000 diluted; Invitrogen), and 5 µl of cDNA. Reaction mixtures were preheated at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Emitted fluorescence was detected online using an SDS7700 real-time PCR system (Applied Biosystems). Gene-specific primers that span one intron were used: ST8SiaII, 5'-GGCTGTGGCCAGGAGATTG-3' and 5'-GGCATACTCCTGAACTGGAGCC-3'; and ST8SiaIV, 5'-GCACCAAGAGACGCAACTCATC-3' and 5'-CAGAGCTGTTGACAAGTGATCTGC-3'. For normalization, primers specific for hypoxanthine-guanine phosphoribosyltransferase were used (5'-TTCCTCATGGACTGATTATGGACA-3' and 5'-AGAGGGCCACAATGTGATGG-3'), and for all primer pairs, the amplification efficiencies were determined by analyzing the slope of a Ct/log (template concentration) plot (30).

DMB-HPLC Analysis—To analyze the chain length and the amount of polySia, P1 mouse brains (80-100 mg) were homogenized, delipidated (31, 32), and dried in a SpeedVac concentrator. Samples were dissolved in 300 µl of DMB reaction buffer and incubated for 24 h at 4 °C with shaking (33). After removal of insoluble material, the reaction was stopped by the addition of 70 µl of 1 M NaOH to 280 µl of supernatant. For separation of polySia chains, we used an LKB HPLC system equipped with a DNAPac PA100 column (Dionex) and a fluorescence detector set at 372 nm for excitation and 456 nm for emission. Milli-Q water and 1 M NaNO3 (E2) were used as eluents at a flow rate of 1 ml/min. Elution was performed by the following gradient: T0 min = 0% (v/v) E2, T5 min = 1% (v/v) E2, T15 min = 10% (v/v) E2, and T60 min = 50% (v/v) E2. The column was washed with 100% (v/v) E2 for 10 min (34). Aliquots of 30 and 300 µl of the supernatants were injected for quantification of peak areas and determination of the maximal detectable chain length, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluation of Methods for PolySia Release—To determine the degree of polymerization (DP), individual polySia chains must be released from NCAM. We applied the DMB-HPLC method developed by Kitajima and co-workers (35) and Inoue and Inoue (31). With this method, polySia chains are released by acid hydrolysis and derivatized in situ with DMB for sensitive fluorometric detection (35). Although the applied conditions preferentially cleave the {alpha}2,3-linkage that attaches polySia to the core glycan, partial hydrolysis of inter-residue {alpha}2,8-sialyl bonds cannot be avoided because of the applied acidic conditions (32, 36).


Figure 1
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  		FIGURE 1.
Chromatographic profiles of polySia from different genotypes. Delipidated brain homogenates obtained from P1 wild-type (A), ST8SiaIV knock-out (B), or ST8SiaII knock-out (C) mice were directly derivatized with DMB and separated on a DNAPac PA100 column. In each case, 9% of the total brain homogenate (equivalent to 7.2 mg of original brain tissue) was injected. To determine the maximally detectable chain length, respective profiles were also generated with 86% aliquots (equivalent to 68.8 mg of brain tissue) as shown in the insets. Because of the marked larger amount of material injected, retention times slightly changed in the latter cases. The number of polySia residues is given for selected peaks. The elution positions of oligomers with DP = 5 were verified with purified sialic acid pentamers. Because brain homogenates were directly labeled with DMB, interfering peaks appeared during the initial phase of chromatography, but did not affect the resolution of polymers with DP ≥ 5 (34). Delipidated brain homogenates obtained from double knock-out (ST8SiaII-/- ST8SiaIV-/-) P1 mice (D) were analyzed as described above, and a 9% aliquot (equivalent to 7.2 mg of original brain tissue) was injected. The inset shows the enlarged profile.

 
While our study was in progress, Nakata and Troy (37) reported an alternative approach for polySia release that avoids production of polySia breakdown products. In their study, the authors used endo-beta-galactosidase to isolate polySia from soluble NCAM, which was polysialylated in vitro by recombinant polysialyltransferases. Because this method yielded significant amounts of polySia from the murine neuroblastoma cell line Neuro2A, we tried to use endo-beta-galactosidase to release polySia chains also from P1 mouse brain samples. However, no release of polySia could be detected. This result confirms published data showing that endo-beta-galactosidase requires di- or poly-N-acetyllactosamine stretches for cleavage (38, 39), which are not found in polysialylated N-glycans of perinatal mouse brain (13, 40).


Figure 2
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  		FIGURE 2.
Alterations in total polySia levels and relative proportions of individual polySia chains. A, brain homogenates (9% aliquots) obtained from wild-type (wt) and mutant mice were analyzed as described in the legend to Fig. 1. Peak areas corresponding to the relative amount of each polySia chain with DP ≥ 8 were calculated and summarized to obtain the total amount of polySia in P1 brains of wild-type and single knock-out mice. Values are the means ± S.D. of five independent experiments, and values obtained for wild-type mice were set to 100%. B, polySia species with chain lengths of 8-20, 21-35, and >35 residues were grouped separately and compared with the respective wild-type compounds. The total amount of polySia in the wild-type mice was set to 100%. C, to quantify the amount of individual polymer sizes, HPLC profiles obtained with 9% aliquots of brain homogenates were used to calculate the peak areas corresponding to the amount of each polySia chain with DP ≥ 8. Values obtained for wild-type mice were set to 100%. Values are the means ± S.D. of five independent experiments. For the sake of clarity, S.D. values registered in the case of wild-type mice are indicated in only D. D, a control experiment was carried out with brain extracts containing defined levels of polySia. Before derivatization with DMB, homogenates obtained from P1 wild-type mouse brains were mixed with brain extracts from polySia-negative double knock-out mice to obtain homogenates containing only 80 and 50% of the wild-type material. The relative amounts of individual polymer sizes were calculated as described above.

 
Length of PolySia Chains Synthesized in Wild-type Mice and Single ST8SiaII or ST8SiaIV Knock-out Mice—To analyze the chain length of polySia synthesized by a combination of ST8SiaII and ST8SiaIV and by each enzyme individually, homogenates obtained from P1 brains of three different genotypes (ST8SiaII+/+ ST8SiaIV+/+, ST8SiaII+/+ ST8SiaIV-/-, and ST8SiaII-/- ST8SiaIV+/+) were used. After DMB derivatization, fluorescently labeled polySia species were separated according to DP by anion-exchange HPLC (32). As discussed above, the partial cleavage of inter-residue sialyl linkages caused typical ladder-like chromatographic profiles for all three genotypes (Fig. 1, A-C). However, in contrast to in vitro studies showing that cooperation between ST8SiaII and ST8SiaIV markedly increases polymer length (41), no significant differences in polySia chain length were detectable in vivo. The maximal chain length (determined in five independent experiments for each genotype) was calculated to be 57 ± 1.6 residues for wild-type mice, 55 ± 1.1 residues for ST8SiaIV knock-out mice, and 53 ± 1.9 residues for ST8SiaII knock-out mice. In ST8SiaII-/- ST8SiaIV-/- mice additionally analyzed by the DMB method, only sialic acid oligomers of up to 7 residues were detected (Fig. 1D). Because no polySia was detectable even if a 10-fold higher amount of homogenate was injected (data not shown), these data unequivocally show that ST8SiaII and ST8SiaIV are the only enzymes synthesizing polySia with DP ≥ 8.

PolySia Levels and Chain Length Distributions in Brain Samples of Single Knock-out Mice Lacking Either ST8SiaII or ST8SiaIV—As indicated by the peak sizes in Fig. 1, the amount of polySia in ST8SiaII-depleted mice was markedly reduced. To quantify polySia in brains of wild-type and mutant mice, the total amount was calculated by adding up all peak areas of a given elution profile, starting with peaks corresponding to oligomers with DP ≥ 8. The results are shown in Fig. 2A. Whereas lack of ST8SiaIV did not change the total amount of polySia, ST8SiaII deficiency caused a reduction to 61% of the wild-type level. To visualize how changes in the polySia amount relate to different polymer sizes, the relative abundance of each polymer size was calculated. Therefore, individual peak areas were determined and compared with wild-type values, which were set to 100% (Fig. 2C). This plot reveals that lack of ST8SiaII led to a decrease in all polymer sizes. However, with increasing polymer length, the reduction became more pronounced, and polySia with DP > 35 was reduced to 30% of the wild-type level (Table 1). As shown in Fig. 2C, also ST8SiaIV deficiency caused a reduction in the amount of long polySia chains.


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  		TABLE 1
Effect of changes in ST8SiaII and ST8SiaIV allele numbers on the polySia pattern in P1 mouse brains

 
In the HPLC profiles (Fig. 1, A-C), peak areas corresponding to polySia chains of >35 residues compose <2% of the total peak areas (Fig. 2B). An overall reduction in polySia may therefore result in loss of the smallest peaks because of the detection limit of the applied method. To test this possibility, we analyzed wild-type brain extracts after dilution with increasing amounts of polySia-negative extracts obtained from double knock-out mice (Fig. 2D). In this control experiment, reduction of the polySia level did not lead to disproportionately low amounts of long chains, and all polymer sizes uniformly decreased according to the percentage of added polySia-negative homogenate. This demonstrates that the sensitivity of the applied HPLC method is sufficient to monitor alterations in the chain length distribution.

NCAM Polysialylation Profiles of ST8SiaII- and ST8SiaIV-deficient Mice—Next, we analyzed the effect of polysialyltransferase deficiency on the NCAM expression level and polysialylation status. We extended our study to mice with all possible combinations of polysialyltransferase allele numbers. A total of nine different genotypes, including wild-type and double knock-out mice, were obtained by appropriate intercrossings, and lysates of P1 brain specimens were prepared and used for Western blotting. The results are displayed in Fig. 3. With mAb 735 (specific for {alpha}2,8-linked polySia with DP ≥ 8) (28, 42), a broad signal above 250 kDa typical for polysialylated NCAM (Fig. 3A, upper panel) was detectable in all genotypes except those for double knock-out animals. In accordance with the lack of polySia demonstrated by DMB-HPLC analysis (Fig. 1D), the latter sample did not contain polySia. A more detailed picture was obtained by staining with anti-NCAM mAb KD11, which detects polysialylated and non-polysialylated NCAM (Fig. 3A, middle panel). In wild-type brain, the exclusive detection of a signal above 250 kDa demonstrated that, at P1, all expressed NCAM was in the polysialylated state. By contrast, only non-polysialylated NCAM was detected in double knockout mice as indicated by two focused bands corresponding to NCAM-180 and NCAM-140, the predominant NCAM isoforms at P1 (43). In heterozygous (ST8SiaII+/- ST8SiaIV+/+, ST8SiaII+/+ ST8SiaIV+/-, and ST8SiaII+/- ST8SiaIV+/-) and ST8SiaIV-null (ST8SiaII+/+ ST8SiaIV-/-) animals, the NCAM pool was almost completely polysialylated, with <6% of the total NCAM in the non-polysialylated state (Fig. 3, A and C). In ST8SiaII-deficient mice, however, 45% of the expressed NCAM was polySia-free (Fig. 3C). This striking difference between mice lacking ST8SiaII or ST8SiaIV was even more pronounced in genotypes with only one functional polysialyltransferase allele. Whereas in ST8SiaII+/- ST8SiaIV-/- mice, only 6% of the brain NCAM pool was non-polysialylated, this fraction increased to 71% in ST8SiaII-/- ST8SiaIV+/- animals (Fig. 3, A and C).

Removal of polySia by treatment of the brain lysates with endosialidase revealed that, in all genotypes, the NCAM isoform pattern (Fig. 3A, lower panel) and protein levels (Fig. 3D) were similar, indicating that polysialyltransferase deficiency does not affect NCAM expression, but instead the percentage of NCAM expressed in the polysialylated state. This effect was most pronounced in mice with a deleted ST8SiaII gene. Only 55 and 34% of the NCAM pool were polysialylated in ST8SiaII-/- ST8SiaIV+/+ and ST8SiaII-/- ST8SiaIV+/- mice, respectively (Fig. 3B). However, in these mutants, the ratio between the measured concentrations of polySia (Fig. 3B) and polysialylated NCAM remained (as in the wild-type mice) close to 1 (Table 1). This leads to the conclusion that, in these genotypes, the amount of polySia added to a single NCAM molecule is identical to the amount bound to NCAM in wild-type mice. By contrast, in ST8SiaII+/- ST8SiaIV-/- mice, the fraction of polysialylated NCAM remained close to 100%, whereas the total polySia level reached only 70% of the wild-type level, thus indicating that polysialylated NCAM carries, on average, 30% less polySia than found in wild-type brain.

PolySia Distribution in Postnatal Mouse Brains of ST8SiaII- and ST8SiaIV-deficient Mice—To investigate whether the marked reduction of polySia in ST8SiaII-deficient mice is due to loss of polySia in particular brain areas or to a general decrease in polySia expression, we compared the spatial distribution of polySia in brains of P1 wild-type and ST8SiaII and ST8SiaIV knock-out mice. Sets of serial coronal and sagittal brain sections were stained with polySia-specific mAb 735, and the results are shown in Fig. 4. In accordance with published data (11, 44), polySia was found almost ubiquitously in brains of P1 wild-type mice (Fig. 4, A-E, left panels). Hippocampal formation and the fimbria, together with the adjacent thalamic and cortical regions, were polySia-immunoreactive throughout (Fig. 4B). In the cortex, only the ventricular zone was devoid of staining, whereas immunoreactivity was strong in the marginal zone and moderate in the cortical plate and intermediate zone (Fig. 4C). Except for the external granular layer, the whole cerebellum exhibited strong polySia expression (Fig. 4, D and E). In both knock-out strains, exactly the same ubiquitous polySia expression pattern as in the wild-type brain was found (Fig. 4, A-E, middle and right panels), demonstrating that the substantial reduction of polySia in ST8SiaII-deficient mice is caused by a general decrease in polySia synthesis and is not associated with loss of polySia in a particular region of the brain.


Figure 3
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  		FIGURE 3.
Polysialylation status of NCAM in wild-type and polysialyltransferase-deficient mice. A, brain lysates of P1 mice of the indicated genotypes were separated by 7% SDS-PAGE using 40 µg of total protein/lane. After transfer to nitrocellulose membrane, proteins were stained with either anti-polySia mAb 735 (upper panel) or anti-NCAM mAb KD11 (middle and lower panels). In parallel experiments, aliquots of each brain lysate were pretreated with endosialidase (endoN) to remove polySia and to investigate total NCAM expression levels (lower panel). Bands representing polysialylated NCAM (polySia-NCAM) and non-polysialylated NCAM-140 and NCAM-180 are indicated on the right. B, shown is a comparison of the total polySia levels (gray bars) and the fraction of polysialylated NCAM (black bars) in P1 mouse brains of the indicated genotypes. Total polySia levels were determined by DMB-HPLC analysis as described in the legend to Fig. 2A. Data represent the means ± S.D. of five independent experiments, and values obtained for wild-type mice were set to 100%. The amount of polysialylated NCAM is given as the difference between the total NCAM expression level and the amount of non-polysialylated NCAM as determined in C and D. C, the relative amount of non-polysialylated NCAM in P1 brains was determined by densitometric evaluation of Western blots stained with anti-NCAM mAb KD11. For each genotype, the intensities of the focused bands at 180 and 140 kDa as shown in the middle panel of A were summed. In each blot, values obtained for the double knock-out mice were used as a reference and set to 100%. Values are the means ± S.D. of four independent experiments. D, the total amount of NCAM expressed in P1 brains was determined by densitometric evaluation of Western blots with endosialidase-treated homogenates. After staining with anti-NCAM mAb KD11 as shown in the lower panel of A, the intensities of both NCAM bands were determined for each genotype on appropriately exposed films. The signals of the wild-type mice were used as a reference in each blot and set to 100%. Data represent the means ± S.D. of four independent experiments.

 
Relative mRNA Levels of ST8SiaII and ST8SiaIV in P1 Brains—To investigate whether loss of one polysialyltransferase gene is compensated by an increased mRNA level of the second enzyme, we determined the relative ST8SiaII and ST8SiaIV transcript levels in P1 brain samples of all genotypes by quantitative real-time RT-PCR (Fig. 5). In the absence of both alleles, no transcript was detected, whereas loss of one allele of either ST8SiaII or ST8SiaIV resulted in a reduction in the corresponding mRNA level by ~50%. This gene dose-dependent reduction in mRNA levels was found for all heterozygous genotypes, irrespective of the presence or absence of the second polysialyltransferase gene. Thus, loss of one allele was not compensated by increased transcription of the remaining allele, indicating independent gene regulation.

Because loss of ST8SiaII caused more severe alterations in the polySia pattern than ST8SiaIV deficiency, we asked whether this is due to marked differences in transcript levels. For direct comparison of mRNA levels of different genes by PCR, it is a prerequisite that target genes are amplified with similar efficiency. Therefore, amplification efficiencies were determined for all primer pairs and turned out to be close to 2 (for details see "Experimental Procedures"). As determined by quantitative real-time RT-PCR, P1 wild-type mouse brains contained only a 2-fold higher transcript level of ST8SiaII compared with ST8SiaIV (1.93 ± 0.58-fold, mean ± S.D. for five independent measurements performed in triplicates). These quantitative data revise earlier studies based on Northern blotting that suggested manifold higher mRNA levels of ST8SiaII compared with ST8SiaIV (16-18).


Figure 4
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  		FIGURE 4.
Comparison of polySia distribution in P1 brains of wild-type, ST8SiaII-deficient, and ST8SiaIV-deficient mice. Sagittal sections of whole brain (A) and coronal sections of the hippocampus (B), cortex (C), and cerebellum (D and E) were stained with anti-polySia mAb 735 before or after removal of polySia by treatment with endosialidase (endoN). Cx, cortex; Cb, cerebellum; Hp, hippocampus; CA, Ammon horn; DG, dentate gyrus; F, fimbria; M, marginal zone; CP, cortical plate; I, intermediate zone; V, ventricle; EG, external granular layer; P, Purkinje cell layer. Scale bars = 2000 µm (A), 350 µm (B-D), and 50 µm (E). Boxes in D indicate areas shown with higher magnification in E.

 


Figure 5
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  		FIGURE 5.
Relative transcript levels of ST8SiaII and ST8SiaIV. The relative mRNA levels of ST8SiaII (upper panel) and ST8SiaIV (lower panel) were determined in P1 brains by quantitative real-time RT-PCR. For each genotype, two independently generated cDNAs were measured in triplicates. Data are the means ± S.D., and values obtained for wild-type mice were set to 100%.

 
Gene Dose Effects of ST8SiaII and ST8SiaIV on the PolySia Chain Length Distribution—The newly acquired knowledge that ST8SiaII and ST8SiaIV transcript levels strictly correlate with the number of functional alleles prompted the question of whether differences in polysialyltransferase mRNA levels affect the polysialylation profile. Therefore, we applied DMB-HPLC analysis to brain samples isolated from P1 heterozygous mice and animals expressing only one functional polysialyltransferase allele. For the sake of clarity, the genotypes will be indicated by the number of polysialyltransferase alleles, starting with the number of ST8SiaII alleles, e.g. 2/0 for ST8SiaII+/+ ST8SiaIV-/- mice (Table 1). Compared with the wild-type genotype (2/2), loss of one ST8SiaII allele (1/2) caused a marked reduction in the amount of polySia chains with DP > 36 (Fig. 6A). Remarkably, however, the observed gene dose-dependent alterations were different in the presence or absence of ST8SiaIV. Lowering the allele number of ST8SiaII from 2 to 1 on the ST8SiaIV-null background (2/0 versus 1/0) resulted in a reduction in the total polySia level from 103 to 70% (Table 1), and alterations affected all polymer sizes (Fig. 6B).

As already shown in Figs. 2A and 3A, ST8SiaIV deficiency was much better compensated by ST8SiaII than vice versa. Consequently, gene dose-dependent alterations for ST8SiaIV were detectable only on the ST8SiaII-null background (Fig. 6, C and D). Interestingly, the observed alterations were restricted to changes in the polySia level and did not affect the chain length distribution as reflected by similar slopes of the two curves in Fig. 6D.

PolySia Chain Length Distribution in Monoallelic Genotypes—As for the single knock-out mice, differences in the total amount of polySia were observed by DMB-HPLC analysis in brain lysates of mice with only one allele of a polysialyltransferase gene (1/0 versus 0/1) (Fig. 7A). In these genotypes, 70% (1/0) and 35% (0/1) of the wild-type polySia level were found (Table 1). In contrast to the distinct differences in the chain length distribution observed between the two single knock-out mice (2/0 versus 0/2) (Fig. 2C), no such effect was seen for the monoallelic genotypes (1/0 versus 0/1) as indicated by comparable curve shapes in Fig. 7A. Comparison of the single knockout (0/2 and 2/0) and monoallelic (0/1 and 1/0) genotypes indicated that the individual mRNA levels play an important role in defining the chain length pattern.

Differences in the Amount of PolySia/NCAM Molecule—Comparison of ST8SiaII-/- ST8SiaIV+/+ and ST8SiaII+/- ST8SiaIV-/- mice (0/2 versus 1/0) demonstrated that these very different genotypes have similar total polySia levels (Fig. 3B), total NCAM levels (Fig. 3D), and comparable chain length patterns (Fig. 7B). However, the two genotypes are clearly different in the percentage of NCAM that is expressed in the polysialylated state (55 and 106% in 0/2 and 1/0, respectively) (Table 1). Thus, in ST8SiaII-/- ST8SiaIV+/+ mice, the same amount of polySia is attached to only one-half of the NCAM pool. The only explanation for this effect is the presence of more polymer chains/NCAM molecule in mice with two alleles of ST8SiaIV compared with mice with one allele of ST8SiaII (0/2 versus 1/0).


Figure 6
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  		FIGURE 6.
Gene dose effect of ST8SiaII and ST8SiaIV on the polySia chain length distribution. P1 brains with the indicated number of polysialyltransferase alleles were analyzed by DMB-HPLC as described in the legend to Fig. 2C, and the amount of each polySia chain in the range of 8-47 residues is given as the means ± S.D. of five independent experiments. Loss of one functional allele of ST8SiaII resulted in stronger changes in the chain length distribution when analyzed on the wild-type (A) or ST8SiaIV-null (B) background. For ST8SiaIV, no significant alteration in the chain length distribution was observed in the presence of ST8SiaII (C), whereas in the absence of ST8SiaII, a clear gene dose-dependent decrease in the amount of polySia was detected for all polymer lengths (D). In brains of mice possessing only one functional ST8SiaII allele (1/0) and ST8SiaII-null mice (0/2), the amounts of polySia with >45 residues were too low for quantification. This was also the case for polySia with >42 residues in ST8SiaII-/- ST8SiaIV+/- (0/1) mice.

 
Polysialylation Patterns Depend on Defined Levels of ST8SiaII and ST8SiaIV—On the basis of the above results, we finally asked if the balance of ST8SiaII and ST8SiaIV expression determines the structural characteristics of synthesized polySia. Brain samples of P1 double heterozygous mice (ST8SiaII+/- ST8SiaIV+/-) where used in this study. As shown in Fig. 3A, almost all expressed NCAM is polysialylated in this genotype, but the chain length pattern clearly differs from that of the wild-type genotype and from the patterns found in the two single knock-out strains (Fig. 7C). This comparison showed that the balanced ratio of ST8SiaII and ST8SiaIV alleles is not sufficient to generate the wild-type pattern, which consequently depends on defined mRNA levels of both enzymes.

All genotypes analyzed are compared in Fig. 7D, which shows similar curve shapes for double heterozygotes and for mice with only one disrupted ST8SiaII allele (1/1 versus 1/2). In the latter genotype, a marked increase in the total amount of polySia (22%) was detected (Table 1), caused by higher abundance of all chain length species. Thus, the addition of one allele of ST8SiaIV (1/1 versus 1/2) affected only the total amount of polySia, but did not alter the chain length distribution. This confirmed that, on average, more polySia chains/NCAM molecule were transferred. However, the overall polymer length remained unchanged. Doubling the ST8SiaII gene dose from double heterozygous to ST8SiaII+/+ ST8SiaIV+/- (1/1 to 2/1) resulted in an increase in the polySia level of 17% and, in addition, led to a drastic change in the chain length distribution, characterized predominantly by an increase in the abundance of polySia with DP > 35. Last but not least, Fig. 7D reveals that, with a single exception (ST8SiaII+/+ ST8SiaIV+/-), the chain length pattern of all mutant mice differed from that of wild-type mice. Interestingly, in none of the mutants was a complete lack of long polymers observed, although the amount of this fraction was predominantly affected by reduced polysialyltransferase gene dosage.


Figure 7
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  		FIGURE 7.
Comparison of the polySia chain length distribution in mutant mice with various combinations of functional polysialyltransferase alleles. P1 brains with the indicated number of polysialyltransferase alleles were analyzed by DMB-HPLC as described in the legend to Fig. 2C, and the amount of each polySia chain in the range of 8-47 residues is given as the means ± S.D. of five independent experiments. Values obtained for the respective wild-type chain length species were set to 100%. A, shown is a comparison of the chain length distribution observed in the two genotypes with only one functional allele of a polysialyltransferase. B, similar chain length distributions were found in ST8SiaII-null mice (0/2) and ST8SiaII+/- ST8SiaIV-/- mice with only one functional allele of ST8SiaII (1/0). C, the polySia chain length distribution in double heterozygous mice (ST8SiaII+/- ST8SiaIV+/-) is compared with the pattern found in wild-type (2/2) and single knock-out (0/2 and 2/0) mice. D, the chain length distributions observed for all genotypes analyzed are summarized in one plot. For the sake of clarity, no S.D. values are given in this representation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PolySia plays an essential role during mouse development as impressively highlighted by the significant phenotypes of mice lacking one polysialyltransferase gene (24, 25) and by the postnatal lethality of mice completely lacking this unique carbohydrate polymer due to ablation of both polysialyltransferase genes (ST8SiaII-/- ST8SiaIV-/-) (26). However, the contribution of each enzyme to the quantity and quality of polySia in vivo has not been addressed so far. Using a panel of eight mutant mice differing only in the number of functional polysialyltransferase alleles, we have demonstrated that alterations in the expression of ST8SiaII and ST8SiaIV affect (i) the total amount of polySia, (ii) the chain length distribution, (iii) the ratio of polysialylated to non-polysialylated NCAM, and (iv) the amount of polySia per NCAM molecule.

The maximal DP of 57 observed in brains of P1 wild-type mice is in good agreement with the chain length of 50-60 residues described previously for polySia in embryonic chicken and postnatal rat brains (34). However, Nakata and Troy (37) reported the presence of small amounts of polySia with DPs of up to ~400 in Neuro2A cells stably transfected with ST8SiaIV using endo-beta-galactosidase for enzymatic release of polySia. Further studies are required to investigate whether such long polymers are restricted to cellular systems overexpressing polysialyltransferases or whether more sensitive methods are required for the detection of polySia chains directly from mouse brain.

We have demonstrated that, in vivo, each enzyme is individually able to synthesize polySia with DP > 50, in contrast to in vitro data showing that ST8SiaII synthesizes shorter polymers than does ST8SiaIV (21, 22). In vitro, both enzymes together yield a higher degree of polysialylation than does either enzyme alone (21, 23). This was not confirmed in vivo because, in wildtype mice, both enzymes together did not yield significantly longer polymers, and the total polySia level was not higher than the sum of polySia found in the two single knock-out mice. Most important, although independent in vitro studies identified ST8SiaII to be less efficient in polySia synthesis compared with ST8SiaIV (12, 21, 22, 41), the most striking changes in NCAM polysialylation in vivo were found to be due to ST8SiaII deficiency. Lack of ST8SiaIV did not change the total polySia level, whereas loss of ST8SiaII resulted in a reduction by 39% accompanied by an altered chain length distribution and an increase in non-polysialylated NCAM from 0 to 45%. This different outcome of in vitro and in vivo analyses convincingly documents that proper enzyme functions crucially depend on the cellular environment. For instance, all in vitro studies were performed with soluble enzymes lacking the transmembrane domain required for Golgi targeting. Because NCAM is also anchored to the Golgi membrane during its transport to the cell surface, fixation of enzyme and acceptor in close proximity might impact processivity and other kinetic properties of the polysialyltransferases.

In line with the largely overlapping expression patterns of ST8SiaII and ST8SiaIV found in murine and rat brains at embryonic day 15 (18, 19), the loss of one polysialyltransferase did not alter the spatial expression pattern of polySia in brains of newborn mice. Consequently, the large fraction of non-polysialylated NCAM (45% of the total NCAM) found in ST8SiaII-deficient brain cannot be attributed to the lack of ST8SiaII in a specific brain area. In ST8SiaII-/- ST8SiaIV+/- mice, the amount of polySia-free NCAM was further increased to 70%, whereas in the presence of a single allele of ST8SiaII (ST8SiaII+/- ST8SiaIV-/-), only 6% of the brain NCAM was devoid of polySia. This remarkable imbalance in the ability of the two enzymes to modify the complete NCAM pool can be due to different enzyme levels or kinetic properties. Northern blot analyses indicated a tremendously higher mRNA level of ST8SiaII in the perinatal phase (16-18). However, as already discussed by Ong et al. (18), ST8SiaII transcript levels determined by Northern blotting or in situ hybridization might be overestimated because, compared with ST8SiaIV-specific probes, ST8SiaII-specific probes have a higher GC content, which accounts for stronger hybridization signals. Using quantitative real-time RT-PCR, we determined that, in P1 wild-type brain, the ST8SiaII transcript level was only 2-fold higher than the ST8SiaIV transcript level. Moreover, the genetic approach demonstrated that, even in the presence of a single ST8SiaII allele (ST8SiaII+/- ST8SiaIV-/-), ~50% more NCAM was polysialylated than in the presence of two ST8SiaIV alleles (ST8SiaII-/- ST8SiaIV+/+), although similar total polysialyltransferase mRNA levels were found in both genotypes. This result indicates that the different NCAM polysialylation capacities of ST8SiaII and ST8SiaIV are due to different kinetic properties of the two enzymes. In line with our in vivo data showing a higher NCAM polysialylation capacity for ST8SiaII, in vitro studies have shown a 3-fold lower NCAM Km value for ST8SiaII compared with ST8SiaIV (22).

The fact that the individual polySia levels observed in both single knock-out strains add up to 164% of the wild-type level (see Table 1) implies that each polysialyltransferase can compensate to a certain extent for the loss of the other enzyme, and our results show that ST8SiaII is more efficient in compensating for the loss of ST8SiaIV than vice versa. The latter observation supports in vitro data demonstrating that ST8SiaIV acts more efficiently on NCAM that already contains polysialic acid on one of the N-glycan antennae and that ST8SiaIV can elongate oligosialic acid synthesized by ST8SiaII (21). Accordingly, loss of ST8SiaII should have a greater impact on the efficiency of ST8SiaIV than vice versa, which is in accordance with our in vivo observation.

The finding that both enzymes can partially compensate for each other is reflected by the mild phenotype of mice lacking only one polysialyltransferase (24, 25). Overlapping functions can be observed in the rostral migratory stream where polysialylated neuronal precursor cells migrate toward the olfactory bulb. In both single knock-out mice, this structure retains polySia expression and is morphologically unaltered (24, 25). Only the complete loss of polySia as in double knock-out mice (ST8SiaII-/- ST8SiaIV-/-) disintegrates the migratory pathway, leading to size reduction of the olfactory bulb (26). In addition to overlapping functions, ST8SiaII and ST8SiaIV also have selective functions that may explain the different phenotype of the single knock-out mice. Loss of ST8SiaII results in defasciculated mossy fibers and ectopic synaptogenesis in the hippocampus (24), morphologic defects that arise during the early postnatal period. Interestingly, the affected fibers are still polySia-positive, but the residual polysialylation achieved by ST8SiaIV does not prevent misrouting of the axons. In line with the observation that ST8SiaII deficiency is not fully compensated by ST8SiaIV, our biochemical analysis of perinatal brain revealed significant alterations of NCAM polysialylation in ST8SiaII-null mice (reduction of polySia by 39%, an altered chain length distribution, and 45% of the NCAM pool shifted to the non-polysialylated state). By contrast, ST8SiaIV deficiency is almost completely compensated by ST8SiaII (no change in the polySia level and the complete NCAM pool remains in the polysialylated state). Only in adult mice, in which ST8SiaII and ST8SiaIV display differential expression patterns, does loss of ST8SiaIV cause a phenotype that is associated with altered synaptic plasticity in the CA1 region of the hippocampus (25). In 6-monthold ST8SiaIV-/- animals, this region is devoid of polySia (25), indicating that ST8SiaII is not expressed at this time point. The differential expression of the two enzymes indicates that the distinct polysialylation patterns produced by each polysialyltransferase are essential for proper brain function.

As shown in this study, changes in the levels of ST8SiaII and ST8SiaIV provide the basis for dynamic alterations in the chain length distribution and the ratio of polysialylated to non-polysialylated NCAM. The polymer dimensions define steric and electrostatic repulsive forces affecting homophilic NCAM-NCAM interactions, but also other cell-surface receptors (3, 4). Consequently, the functional role of polySia may vary according to chain length, resulting in flexible regulation of cell interactions. Because polySia forms a large hydrated structure, which can extend beyond the protein core (45), the polymer size and number of polySia chains/NCAM molecule will determine the accessibility of NCAM for homo- and heterophilic interactions and thereby affect cell signaling (26, 46). Thus, precise adjustment of polysialylation levels might be essential for the fine-tuning of NCAM-mediated interactions. Understanding the individual impact of the two polysialyltransferases ST8SiaII and ST8SiaIV in vivo provides the basis to unravel the mechanisms controlling the polySia pattern during development, plasticity, and aging of the nervous system, but also during the progression of polySia-expressing tumors (46), as well as in neurodegenerative and neuropsychiatric disorders (47-50).


   FOOTNOTES
 
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to H. G., R. G., R. G.-S., and M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: 49-511-532-9807; Fax: 49-511-532-3956; E-mail: muehlenhoff.martina{at}mh-hannover.de.

2 The abbreviations used are: polySia, polysialic acid; NCAM, neural cell adhesion molecule; P1, postnatal day 1; mAb, monoclonal antibody; DMB, 1,2-diamino-4,5-methylenedioxybenzene; RT, reverse transcription; HPLC, high pressure liquid chromatography; DP, degree of polymerization. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jamey Marth for kindly providing ST8SiaII knock-out mice; Dr. Ulrich Lehmann for help with the realtime RT-PCR assays; and Ulrike Peters, Daniela Wittenberg, and Kerstin Leib for excellent technical assistance. We are indebted to Y. and S. Inoue for advice in establishing the DMB-HPLC technique.



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